Diabetes is a major risk factor for cardiovascular disease, culminating in myocardial infarction, and heart failure. Prolonged hyper-0-GlcNAcylation, due to nutrient excess and hyperglycemia, is a major molecular cause of glucose toxicity and insulin resistance. Increased 0-GlcNAcylation directly contributes to diabetic cardiomyopathy and to dysfunctional mitochondria, perhaps contributing to excessive production of reactive oxygen species (ROS). Even though 0-GlcNAcylation clearly plays an important role in diabetic cardiovascular disease, virtually nothing is known about 0-GlcNAcylation in the cardiomyocyte. This project will elucidate the roles of 0-GlcNAc in diabetic cardiomyopathy and will define the 0-GlcNAcome of the cardiomyocyte at the site-specific level. Specific Aims: Aim 1: Quantify the Site-Specific Crosstalk Between 0-GlcNAcylation and Phosphorylation in the cardiomyocyte proteome and in purified cardiomyocyte mitochondria from Normal and Diabetic Rats. Using chemico-enzymatic photocleavable tag enrichment combined with electron transfer dissociation (ETD) tandem mass spectrometry, we will quantify site occupancy for both 0-GlcNAc and phosphate in cardiomyocyte contractile and mitochondrial proteins from normal and diabetic rats. Aim 2: Determine the Specific Roles of 0-GlcNAcylation in normal cardiomyocyte mitochondria, and the sites of action and mechanisms of diabetes-induced dysfunction, leading to ROS production. We wilt specifically alter 0-GlcNAcylation using methods developed during the past 20-years, and correlate alterations with specific mitochondrial function. Aim 3: Elucidate the properties and regulation of cardiomyocyte mitochondrial isoforms of O-GlcNAc Transferase and 0-GlcNAcase. Virtually nothing is known about the mitochondrial isoforms of 0-GlcNAc Transferase (OGT) or 0-GlcNAcase (OGA). We will elucidate their localization, activities, molecular associations and kinetic activities in mitochondria from normal and diabetic rats. Aim 4: Evaluate the affects and roles of diabetes-induced mitochondrial dysfunction and increased O- GlcNAcylation of cardiomyocyte contractile machinery on cardiac physiology and function. Working closely with Core D we will systematically evaluate the importance of the crosstalk between 0-GlcNAcylation and phosphorylation of cardiomyocyte contractile and mitochondrial proteins on the physiological functions of cardiomyocytes These studies will open a new paradigm for understanding the regulation of cardiac functions and in diabetic cardiomyopathies. They will lead to totally unexplored avenues of possible therapeutic interventions RELEVANCE (See instructions): Diabetes is a major epidemic and contributes to cardiovascular disease, which ultimately results in heart failure or myocardial infarction. Increased 0-GlcNAcylation, a sugar post-translational modification, underlies molecular events contributing to diabetic cardiomyopathies by affecting the functions of contractile and mitochondrial proteins within the cardiomyocyte. These studies will elucidate the importance of O- GlcNAc in both normal and diabetic cardiomyocyte physiology, and will possibly lead to novel treatments.